research_2009

Research Interest

Structural studies of nucleic acids-binding proteins in translation regulation and nucleic acid degradation

We are interested in a number of proteins involved in translation regulation and DNA/RNA degradation. The overall goal is to discover structure-based mechanisms of these proteins in nucleic acids recognition and degradation. We use a major tool of X-ray crystallography in combination with mutagenesis, biochemical and biophysical approaches. Several projects of interest are listed below.

1. Bacterial nucleases in cell defense

We have been working on two types of sugar non-specific nucleases in bacteria, including a periplasmic nuclease Vvn and a secreted toxin ColE7, both of which digest foreign nucleic acids for cell defense. Based on our structural and biochemical analysis on Vvn and ColE7, we have provided a solid foundation to explain how these nucleases are inhibited and activated, how they recognize DNA without sequence specificity and how they digest DNA to protect bacterial cells at atomic level.

References:

Li, C., Ho, L.-I., Chang, Z.-F., Tsai, L.-C., Yang, W.-Z. and Yuan*, H. S. (2003) DNA binding and cleavage by the periplasmic nuclease Vvn: A novel structure with a known active site. EMBO J. 22, 4014-4025.

Hsia, K.-C., Chak, K.-F., Liang, P.-H., Cheng, Y.-S., Ku, W.-Y. and Yuan*, H. S. (2004) DNA binding and degradation by the H-N-H protein ColE7. Structure 12, 205-214.

Hsia, K.-C., Li, C.-L. and Yuan*, H. S. (2005) Structural and functional insight into the sugar-nonspecific nucleases in host defense, Curr. Opin. Struct. Biol. 15, 126-134.

Shi, Z., Chak, K.-F. and Yuan*, H. S. (2005) Identification of an essential cleavage site in ColE7 required for import and killing cells, J. Biol. Chem. 26, 24663-24668.

Cheng, Y.-S., Shi, Z., Doudeva, L. G., Yang, W.-Z., Chak, K.-F. and Yuan*, H. S. (2006) High-resolution crystal structure of a truncated ColE7 translocation domain: Implications for colicin transport across membranes. J. Mo. Biol., 356, 22-31.

Wang, Y.-T., Yang, W.-J., Li, C.-L., Doudeva, L. G. and Yuan*, H. S. (2007) Structural basis for sequence-dependent cleavage by nonspecific endonucleases. Nucleic Acid Res. 35, 584-594.

2. Tudor-SN in miRNA degradation and mRNA translation regulation

Tudor-SN is a multifunctional protein, playing a role in transcription regulation, RNA editing, interference and splicing. Recent studies show that Tudor-SN is a miRNase specific for inosine-containing microRNA precursors, and it also regulates gene expression by binding to mRNA at 3’UTR to decrease the rate of mRNA decay. Human Tudor-SN contains five staphylococcal nuclease-like (SN) and a tudor domains. Our structural and biochemical analysis of a truncated 64-kD Tudor-SN shows the architecture and assembly of SN and tudor domains and also suggests that two SN domains work together functioning as a clamp to capture RNA substrates. Co-crystallization (with RNA), biochemical and mutagenesis experiments are underway to further reveal the molecular basis of Tudor-SN in mRNA recognition and miRNA cleavage.

Structural model of a 64-kD Tudor-SN bound to double-stranded RNA.
Our biochemical and structural data suggest that tandem repeats of SN domains in Tudor-SN work together to capture RNA substrates.

References:

Li, C.-L., Yang, W.-Z., Chen, Y.-P. and Yuan*, H. S. (2008) Structural and functional insights into human Tudor-SN, a key component linking RNA interference and editing. Nucleic Acid Res. 36, 3579-3589.

3. PNPase in mRNA degradation

PNPase (polynucleotide phosphorylase) is an important enzyme responsible for mRNA turnover from 3’- to 5’-end in bacteria. It shares a similar structural organization to eukaryotic exosome core complexes. Our structural and biochemical study on E. coli PNPase shows that the trimeric structure of a KH/S1-truncated PNPase is more expanded, containing a slightly wider central RNA-binding channel than that of the wild-type PNPase. This result suggests that the KH/S1 domain is involved not only in RNA binding but it also helps PNPase to assemble into a more compact trimer. This finding is likely a general phenomenon since only bacterial PNPase and archaeal exosomes with constricted channels are efficient enzymes in RNA degradation. We also study the structures and biochemical properties of human exosome component proteins and mitochondrial PNPase to further characterize the structure-and-function relationship of PNPase in mRNA binding and degradation.

Crystal structure of E. coli PNPase. The homotrimeric PNPase is assembled into a ring-like structure which contains a central channel for RNA binding and digestion. Our structural and mutational studies show that the arginine residues located in the central channel play crucial roles in trapping RNA for processive exonucleolytic degradation.

Reference:

Shi, Z., Yang, W.-Z., Lin-Chao, S., Chak, K.-F. and Yuan*, H. S. Crystal structure of Escherichia coli PNPase: central channel residues are involved in processive RNA degradation. RNA (in press).

4. Apoptotic nucleases in DNA degradation.

Apoptotic nucleases are activated for chromosomal DNA fragmentation during apoptosis. Inactivation of these apoptotic nucleases produces undigested DNA and is related to a number of autoimmune disorders. We analyze the biochemical properties and crystal structures of a number of apoptotic nucleases to address the function of these nucleases in normal versus apoptotic cells. Recently, we determined the crystal structure of a C. elegans cell-death-related nuclease 4 (CRN-4). The biochemical, structural, and functional assays consistently suggest that the C-terminal novel-fold Zn-domain of CRN-4 is involved in DNA binding and the N-terminal nuclease domain is responsible for DNA degradation. This study therefore provides new insights into the DEDDh family of nucleases in chromosomal DNA fragmentation in apoptosis.

We also analyze the biochemical and structural features of several apoptotic proteins and nucleases that interact with CRN-4 to form a degradeosome in apoptosis, including CPS-6 (human Endo G homologue), WAH-1 (AIF), CRN-5 (Rrp46) and Cyp-13. The long-term goal of this research is to decipher the working mechanism of the degradeosome in DNA fragmentation during apoptosis.

Crystal structure of CRN-4 and Comparison of the different domain arrangement in dimeic DEDDh family proteins. CRN-4 dimerizes in a different mode as compared to PARN, TREX2 and RNase T.

Reference:

Hsiao, Y. Y., Nakagawa, A., Shi, Z., Mitani, S., Xue^,D. and Yuan*, H. S. Crystal structure of CRN-4: implications for domain function in apoptotic DNA degradation. (under revision).

5. TDP-43 in RNA binding and pathogenic aggregation.

TDP-43 is an important disease protein: its normal function in RNA binding is related to the common lethal genetic disease cystic fibrosis, and its abnormal aggregation in brain cells is directly linked to the neurodegenerative disorders of FTLD (frontotemporal lobar degeneration) and ALS (amyotrophic lateral sclerosis). Our crystal structural determination of TDP-43 RRM2 domain in complex with DNA reveals the basis of its preference for TG/UG-rich sequences. It also reveals an atypical RRM fold for the RRM2 domain with an additional b-strand involved in making non-native protein-protein interactions, facilitating aggregation of TDP-43 into higher order filamentous assemblies. These results provide a testable model for studying non-amyloid aggregates in neurodegenerative diseases related to TDP-43 proteinopathy. More biochemical and structural studies in characterizing the native and aggregated structures of TDP-43 are underway.

Crystal structure of TDP-43 RRM2 domain showing that RRM2 has an extra b-strand (b-4) as compared to a typical RNA recognition motif.

The crystal packing diagram of RRM2-DNA complex. RRM2 assembles into a fiber-like structure with b-strands forming a continuous b-sheet wrapping around the fiber-like helix structure.

Reference:

Kuo, P,-S, Doudeva, L. G., Wang, Y.-T., Shen, C.-K. J. and Yuan*, H. S. (2009) Structural insights into TDP-43 in nucleic acid binding and domain interactions. Nucleic Acids Res. (in press).

Data collection at NSRRC (National Synchrotron Radiation Research Center), Hsin-Chu, Taiwan.

2008 IUCr Congress (Osaka, Japan)